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一种微管-荧光融合蛋白嵌合标记斑马鱼运动神经元系统的建立

袁芳^1,2, 钱佩佩¹, 王新³, 盛佳婧¹, 刘东^1,*, 巩杰¹

¹南通大学生命科学学院，南通 226001；²南通大学医学院，南通 226001；³南通大学神经再生重点实验室，南通 226001

摘要

运动神经元是一类支配运动行为的重要神经元。传统的荧光蛋白标记的转基因斑马鱼品系(用于活体成像分析运动神经元形态发生)存在胞体密集、突触交错、不好区分单个神经元等不足。为了优化活体成像分析运动神经元，本研究旨在建立一种微管-荧光融合蛋白嵌合标记斑马鱼运动神经元系统。首先通过Gateway克隆技术将运动神经元表达基因mnx1启动子序列与绿色荧光蛋白-α-Tubulin融合蛋白序列构建到含有Tol2转座位点的表达载体中，然后将该质粒和Tol2 mRNA同时注射到4~8细胞期斑马鱼受精卵中，在72 hpf (hours post fertilization)进行共聚焦显微成像分析。结果显示，该系统中绿色荧光融合蛋白在3种类型运动神经元中表达，从而实现单个运动神经元嵌合标记。本研究进一步探索注射剂量与嵌合标记神经元数量以及分布频率的关系，并确定了重组蛋白的合适剂量(15 ng)。此外，本研究在该模型上验证了insm1a和kif15表达下调导致的运动神经元异常发育。这些结果表明我们成功建立了一种微管-荧光融合蛋白嵌合标记斑马鱼运动神经元系统，为探究运动神经元的发育和形态发生提供了一个直观和快速的模型，在运动神经元病发病机制和药物筛选等研究中有潜在的应用价值。

关键词： 转基因; 嵌合体; 运动神经元; 斑马鱼

Establishment of a microtubule-fluorescent fusion protein mosaically labeled zebrafish motor neuron system

YUAN Fang^1,2, QIAN Pei-Pei¹, WANG Xin³, SHENG Jia-Jing¹, LIU Dong^1,*, GONG Jie¹

¹School of Life Science, Nantong University, Nantong 226001, China；²School of Medical School, Nantong University, Nantong 226001, China；³Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, China

Abstract

Motor neurons are an important type of neurons that control movement. The transgenic fluorescent protein (FP)-labeled motor neurons of zebrafish line is disadvantageous for studying the morphogenesis of motor neurons. For example, the individual motor neuron is indistinguishable in this transgenic line due to the high density of the motor neurons and the interlaced synapses. In order to optimize the in vivo imaging methods for the analysis of motor neurons, the present study was aimed to establish a microtubule- fluorescent fusion protein mosaic system that can label motor neurons in zebrafish. Firstly, the promotor of mnx1, which was highly expressed in the spinal cord motor neurons, was subcloned into pDestTol2pA2 construct combined with the GFP-α-Tubulin fusion protein sequence by Gateway cloning technique. Then the recombinant constructs were co-injected with transposase mRNA into the 4–8 cell zebrafish embryos. Confocal imaging analysis was performed at 72 hours post fertilization (hpf). The results showed that the GFP fusion protein was expressed in three different types of motor neurons, and individual motor neurons were mosaically labeled. Further, the present study analyzed the correlation between the injection dose and the number and distribution of the mosaically labeled neurons. Fifteen nanograms of the recombinant constructs were suggested as an appropriate injection dose. Also, the defects of the motor neuron caused by the down-regulation of insm1a and kif15 were verified with this system. These results indicate that our novel microtubule-fluorescent fusion protein mosaic system can efficiently label motor neurons in zebrafish, which provides a more effective model for exploring the development and morphogenesis of motor neurons. It may also help to decipher the mechanisms underlying motor neuron disease and can be potentially utilized in drug screening.

Key words: transgenosis; mosaic; motor neuron; zebrafish

收稿日期：　　录用日期：

通讯作者：刘东　　E-mail: liudongtom@gmail.com

DOI: 10.13294/j.aps.2022.0049

引用本文：

袁芳, 钱佩佩, 王新, 盛佳婧, 刘东, 巩杰. 一种微管-荧光融合蛋白嵌合标记斑马鱼运动神经元系统的建立[J]. 生理学报 2022; 74 (3): 411-418.

YUAN Fang, QIAN Pei-Pei, WANG Xin, SHENG Jia-Jing, LIU Dong, GONG Jie. Establishment of a microtubule-fluorescent fusion protein mosaically labeled zebrafish motor neuron system. Acta Physiol Sin 2022; 74 (3): 411-418 (in Chinese with English abstract).